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1.
Liver glycogen synthase activity is increased, and glycogen phosphorylase activity and glucose 6-phosphate content reduced by in vivo insulin during a euglycemic hyperinsulinemic clamp in lean young adult rhesus monkeys. To examine the mechanism of dephosphorylation of liver glycogen synthase and glycogen phosphorylase, the enzyme activities of protein phosphatase-1, protein phosphatase-2C, cAMP-dependent protein kinase, glycogen synthase kinase-3, protein kinase C and protein tyrosine kinase were determined before and after three hours of in vivo insulin in these same monkeys. The bioactivity of an inositol phosphoglycan insulin mediator (pH 2.0) and cAMP concentrations were also measured in the liver before and after insulin administration. Insulin caused significant increases in protein phosphatase-1 (p = 0.005) and in protein phosphatase-2C activities (p = 0.001). Insulin-stimulated minus basal bioactivity of the pH 2.0 insulin mediator was strongly inversely related to the insulin-stimulated minus basal glucose 6-phosphate content (r = -0.93, p < 0.0001). These findings suggest that protein phosphatase-1 and protein phosphatase-2C may be involved in the mechanism of in vivo insulin activation of liver glycogen synthase and inactivation of liver glycogen phosphorylase.  相似文献   

2.
Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.  相似文献   

3.
Glycogen from the thermophilic eubacterium Thermus thermophilus has been characterized by enzymatic, chemical and spectroscopic analysis. With an average chain length of seven glucose units, the glycogen from T. thermophilus is one of the most highly branched glycogens known. In contrast to other bacterial species, in T. thermophilus, accumulation of glycogen appears not be affected by low nitrogen concentration. For the first time, alpha-glucan phosphorylase activity and glycogen content were measured throughout the growth cycle of T. thermophilus in order to gain insight into glycogen metabolism. In contrast to the situation that prevails in Escherichia coli, additional carbon sources had no effect on alpha-glucan phosphorylase activity in T. thermophilus. Maximal activity of the thermophilic enzyme was found in the early logarithmic phase of growth, suggesting a function of the alpha-glucan phosphorylase in T. thermophilus as an outgrowth-specific enzyme.  相似文献   

4.
Relationships between pH and the concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), and lactate during ischemic exercise depend on passive buffering, proton consumption as a consequence of net PCr breakdown, the control of glycogenolysis, (particularly in relation to the concentration of Pi, a substrate of glycogen phosphorylase that is produced by net PCr breakdown), and the creatine kinase equilibrium. The author analyzes the implications of these relationships for the interpretation of 31P-magnetic resonance spectroscopic data and for the control of glycogenolysis. For realistic adenosine diphosphate (ADP) concentrations, given the constraints of the creatine kinase equilibrium, the pH must be near-linear with lactate, with an apparent buffer capacity (i.e., the ratio of lactate accumulation to pH change) that is nearly twice the true buffer capacity (i.e., the ratio of net proton loading to pH change). The implications for glycogenolytic control depend on adenosine triphosphate (ATP) turnover, but an upper limit of activation of glycogen phosphorylase (i.e., the amount of the a form) that would permit no increase in ADP concentration can be calculated. Phosphorylase activation during ischemic exercise seems approximately proportional to the power output, consistent with calcium stimulation of phosphorylase b kinase. In simulations, ADP concentration is highly sensitive to this proportionality, as (unlike in purely oxidative exercise) ADP concentration is not known to participate in any closed feedback loops in ischemic exercise.  相似文献   

5.
With the goal to obtain maltose phosphorylase as a tool to determine ortho-phosphate, the enzyme from Lactobacillus brevis was purified to 98% by an expeditious FPLC-aided procedure which included anion exchange chromatography, gel filtration, and hydroxyapatite chromatography. The native maltose phosphorylase had a molecular mass of 196 kDa and consisted of two 88 kDa subunits. In isoelectric focusing two isoforms with pI values of 4.2 and 4.6 were observed. Maximum enzyme activity was obtained at 36 degrees C and pH 6.5 and was independent of pyridoxal 5'-phosphate. The apparent K(m) values with maltose and phosphate as substrates were 0.9 mmol l-1 and 1.8 mmol l-1, respectively. Maltose phosphorylase could be stored in 10 mM phosphate buffer pH 6.5 at 4 degrees C with a loss of activity of only 7% up to 6 months. The stability of the enzyme at high temperatures was enhanced significantly using additives like phosphate, citrate, and imidazole. The purified maltose phosphorylase was used as key enzyme in a phosphate sensor consisting of maltose phosphorylase and glucose oxidase. A detection limit of 0.1 microM phosphate was observed and the sensor response was linear in the range between 0.5 and 10 microM.  相似文献   

6.
Recent evidence indicates that inflammatory cytokines are involved in changes of blood glucose concentrations and hepatic glucose metabolism in infectious diseases, including sepsis. However, little is known regarding how cytokines interact with glucoregulatory hormones such as insulin. The objective of the present study is to investigate if and how cytokines influence insulin-stimulated glycogen metabolism in the liver. Interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) markedly inhibited the increase of glycogen deposition stimulated by insulin in primary rat hepatocyte cultures; however, tumor necrosis factor alpha had no effect. Labeling experiments revealed that both cytokines counteracted insulin action by decreasing [14C]-glucose incorporation into glycogen and by increasing [14C]-glycogen degradation. Furthermore, it was discovered that IL-1beta and IL-6 inhibited glycogen synthase activity and, in contrast, accelerated glycogen phosphorylase activity. In experiments with kinase inhibitors, serine/threonine kinase inhibitor K252a blocked IL-1beta- and IL-6-induced inhibitions of glycogen deposition, as well as glycogen synthase activity, whereas another kinase inhibitor staurosporine blocked only IL-6-induced inhibition. Tyrosine kinase inhibitor herbimycin A blocked only IL-1beta-induced inhibition. These results indicate that IL-1beta and IL-6 regulate insulin-stimulated glycogen synthesis through different pathways involving protein phosphorylation in hepatocytes. They may mediate the change of hepatic glucose metabolism under pathological and even physiological conditions by modifying insulin action in vivo.  相似文献   

7.
A phosphatase, active towards phosphorylase a and phosphorylated proteins casein and histone II-A, was isolated from Saccharomyces cerevisiae 257. The enzyme dephosphorylated glycogen phosphorylase from commercial yeast rendering it inactive. The protein phosphatase activity was not influenced by any metal ions. Phosphorylase phosphatase activity was slightly stimulated by p-nitrophenyl phosphate and inhibited by heparin.  相似文献   

8.
The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon diameter. Immunocytochemical analyses of spinal cord revealed axonal localization of all protein phosphatase subunits. To determine whether protein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel filtration. approximately 15% of the total phosphorylase a phosphatase activity was present in the neurofilament fraction. The catalytic subunits of PP1 and PP2A, as well as the A and B alpha regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting, whereas PP2B and PP2C were found exclusively in the low molecular weight soluble fractions. PP1 and PP2A subunits could be partially dissociated from neurofilaments by high salt but not by phosphatase inhibitors, indicating that the interaction does not involve the catalytic site. In both neurofilament and soluble fractions, 75% of the phosphatase activity towards exogenous phosphorylase a could be attributed to PP2A, and the remainder to PP1 as shown with specific inhibitors. Neurofilament proteins were phosphorylated in vitro by associated protein kinases which appeared to include protein kinase A, calcium/calmodulin-dependent protein kinase, and heparin-sensitive and -insensitive cofactor-independent kinases. Dephosphorylation of phosphorylated neurofilament subunits was mainly (60%) catalyzed by associated PP2A, with PP1 contributing minor activity (10-20%). These studies suggest that neurofilament-associated PP1 and PP2A play an important role in the regulation of neurofilament phosphorylation.  相似文献   

9.
The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.  相似文献   

10.
In neutrophils, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the translocation of the Ca(++)- and phospholipid-dependent protein kinase, protein kinase C (PK-C) from the soluble to the particulate fraction. At the same time there was a corresponding increase in the amount of Ca(++)- and phospholipid-independent protein kinase activity recovered in the soluble fraction. This soluble Ca(++)- and phospholipid-independent protein kinase presumably reflects proteolytic activation of the particulate associated PK-C. Bone marrow and undifferentiated HL-60 cells also translocated PK-C to the particulate fraction in response to TPA but did not accumulate the soluble Ca(++)- and phospholipid-independent form of the enzyme. Similar results were obtained using HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO), recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) or 1 alpha,25-dihydroxyvitamin D3. There was also no significant change in either the number or time of expression of differentiation-specific cell surface antigens observed on HL-60 cells induced to differentiate with either DMSO, 1 alpha,25-dihydroxyvitamin D3 or TPA in the presence of cyclosporin A, an agent reported to inhibit the proteolytic breakdown of PK-C to the Ca(++)- and phospholipid-independent form. Likewise, cyclosporin A did not affect the rate of extent of differentiation of primary bone marrow cell cultures. These results suggest that the proteolytically activated and phospholipid-independent form of PK-C is probably not involved in haemopoietic cell differentiation.  相似文献   

11.
Alpha-Aminoadipate aminotransferase and kynurenine aminotransferase activities were co-purified from the rat kidney supernatant fraction. The resulting preparation was determined to be nearly homogenous by analytical disc gel electrophoresis at pH 8.9 and 7.5 isoelectric focusing on polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of approximately 85,000 was determined on Sephadex G-200 chromatography and sucrose density gradient analysis. The enzyme was determined to be comprised of two subunits of approximately the same molecular weight (45,500 +/- 850) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An isoelectric pH of 6.56 +/- 0.06 was determined by focusing on polyacrylamide gels. Further evidence is provided to support the idea that the alpha-aminoadipate aminotransferase and kynureine aminotransferase activities are properties of a single protein: (a) co-purification of the two activities from the rat kidney supernatant fraction with the ratio of their specific activities remaining constant, (b) similar chromatographic behavior, (c) a similarity in their dependence on added pyridoxal-P for activity, and (d) a similar pattern of heat inactivation.  相似文献   

12.
In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.  相似文献   

13.
In fetal livers of both man and rat thymidine kinase activity was 12 times higher than in the adult, glutamate dehydrogenase and arginase were present at 20-50% of their adult values, whereas alanine aminotransferase activity was only an insignificant fraction of that in the adult. Although the developmental changes for the four enzymes were quantitatively similar in both species, qualitatively there were some significant differences. In adult human liver, glutamate dehydrogenase activity was distributed almost equally between the cytosol and particles; the concentration of only the soluble enzyme increased after birth. In rat liver, glutamate dehydrogenase remained exclusively a particulate enzyme. The soluble hepatic alanine aminotransferase activity rose in both species after birth (from less than 2 U/g to 41-57 U/g, respectively). Thymidine kinase was wholly soluble in the fetal livers; only in adult human liver was additional activity (at least 50% of the total) found in the particles. Arginase isozymes, identical and apparently the same single isozyme in fetal and adult rat liver, show an ontogenetic change in man. A shift from a single form, common to human fetal liver and fetal kidney, to at least two variants in adult human liver, indicates another complexity of the fully differentiated liver in man.  相似文献   

14.
Gluthathione peroxidase (gluthatione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) has been purified approximately 2700-fold from rat lung soluble fraction. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate/urea polyacrylamide gel electrphoresis. Selenium-75 tracer cochromatographed with the enzyme activity, indicating that rat lung soluble gluthathione peroxidase is a selenium enzyme. The enzyme had an approximate molecular weight of 80000 and contained four identical subunits. The optimal activity of the enzyme was at between pH 8.8 and 9.1. The enzyme had general specificity toward hydroperoxides, and high specificity for reduced glutathione. The kinetic behavior or the purified lung soluble glutathione peroxidase followed a ping-pong-like mechanism; the enzyme first reduced the lipid hydroperoxide substrate to the corresponding hydroxy fatty acid, then was regenerated to the native form by reduced glutathione.  相似文献   

15.
Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report. Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells. Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells. Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively. In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40%. In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all. Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration. Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells. We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin.  相似文献   

16.
The mechanism by which beta adrenergic agonist stimulate glycogenolysis in intact skeletal muscle was investigated in mice with the phosphorylase kinase deficiency mutation (I strain). Although extracts of I strain diaphragm muscle had only 3.7% of the phosphorylase kinase activity found in extracts of the control strain (C57BL), incubation of I strain hemidiaphragms in Krebs-Ringer bicarbonate buffer with either isoproterenol or epinephrine resulted in a stimulation of the rate of glycogenolysis. In C57BL diaphragms, the EC50 values for isoproterenol and epinephrine were 2 and 14 nM, respectively. With I strain diaphragms, dl-isoproterenol or l-epinephrine stimulated glycogenolysis as a linear function of the log of the drug concentration with no apparent plateau of response up to concentrations of 30 to 40 mugM. For each 10-fold increase in drug concentration, isoproterenol and epinephrine stimulated glycogenolysis in I strain muscles an additional 0.37 to 0.42 mg/g/hr, a slope in the concentration-response relationship of 0.17 and 0.37, respectively, of that measured in C57BL diaphragms at concentrations around the EC50. The highest glycogenolytic response measured in I strain hemidiaphragms (at 40 mugM isoproterenol) was 80% of the maximal catecholamine-stimulated glycogenolysis in C57BL diaphragms. Both 4 nM and 4 mugM isoproterenol, in a concentration-dependent manner, stimulated phosphorylase b to a conversion in I and C57BL diaphragms and increased cyclic adenosine 3':5'-monophosphate (cyclic AMP) concentrations. The glycogenolytic response to 10.1 nM dl-isoproterenol in both I and C57BL diaphragms was blocked by 34 nM l-propranolol but not by 34 nM d-propranolol. The response to 4 mugM isoproterenol was enhanced by the cyclic nucleotide phosphodiesterase inhibitors papaverine (27 mugM) or dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 3 mugM). From the results of these studies, we conclude: 1) Catecholamines stimulate glycogenolysis in skeletal muscle of I mice, as in C57BL mice, by interacting with the beta adrenergic receptor, thereby increasing tissue cyclic AMP concentrations and stimulating phosphorylase b to a conversion. 2) alternative hypotheses for the mechanism of the catecholamine-stimulated decrease in glycogen concentration in I skeletal muscle-inhibition of glycogen synthesis, hyposia and 5'-AMP stimulation of phosphorylase b activity-have been ruled out. 3) the activity of the mutant phosphorylase kinase, although it is only 3.7% of that in extracts of C57BL muscle, is sufficient to produce phosphorylase b to a conversion and thereby account for the glycogenolytic response of I strain muscle to catecholamines.  相似文献   

17.
alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase b (Ki = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that, in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several alpha- and beta-anhydroglucoheptonic acid derivatives and 1-deoxy-1-thio-beta-D-glucose analogues have been synthesized and tested in a series of crystallographic and kinetic binding studies with glycogen phosphorylase. The structural results of the bound enzyme-ligand complexes have been analyzed, together with the resulting affinities, in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. This work has shown the following: (i) Similar affinities may be obtained through different sets of interactions. Specifically, in the case of the alpha- and beta-glucose-C-amides, similar Ki's (0.37 and 0.44 mM, respectively) are obtained with the alpha-anomer through interactions from the ligand via water molecules to the protein and with the beta-anomer through direct interaction from the ligand to the protein. Thus, hydrogen bonds through water can contribute binding energy similar to that of hydrogen bonds directly to the protein. (ii) Attempts to improve the inhibition by additional groups did not always lead to the expected result. The addition of nonpolar groups to the alpha-carboxamide resulted in a change in conformation of the pyranose ring from a chair to a skew boat and the consequent loss of favorable hydrogen bonds and increase in the Ki. (iii) The addition of polar groups to the alpha-carboxamide led to compounds with the chair conformation, and in the examples studied, it appears that hydration by a water molecule may provide sufficient stabilization to retain the chair conformation. (iv) The best inhibitor was N-methyl-beta-glucose-C-carboxamide (Ki = 0.16 mM), which showed a 46-fold improvement in Ki from the parent beta-D-glucose. The decrease in Ki may be accounted for by a single hydrogen bond from the amide nitrogen to a main-chain carbonyl oxygen, an increase in entropy through displacement of a water molecule, and favorable van der Waals contacts between the methyl substituent and nonpolar protein residues.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

18.
Glycogen storage diseases   总被引:1,自引:0,他引:1  
Each of 12 types of glycogen storage disease (GSD O-XI) is delineated by clinical, biochemical and histologic features that allow its identification in future patients. GSD II occurs in 2 forms that are not both encountered in the same family. GSD IIa is the infantile fatal form with cardiomegaly, increased cardiac glycogen concentration and cardiac failure; GSD IIb is the adult form with clinically normal heart and normal cardiac glycogen concentration. Nonetheless, the heart muscle of both forms is equally deficient in acid alpha-glucosidase activity, and this raises questions as to the latter's role in the pathophysiology of GSD II. The appearance of hepatocytes in GSD IIa becomes normal after the administration of alpha-glucosidase. Using electron microscopy of uncultured amniotic fluid cells, the prenatal diagnosis of GSD IIa is feasible within one day after the amniocentesis. GSD VI and IX are instances of benign hepatomegaly except when GSD IX and III occur in the same child; one such patient died suddenly at home. There are 2 modes of inheritance in GSD IX: one (GSD IXa) is autosomal recessive, the other one (GSD IXb) is X-linked recessive. In either form the Km of the remaining liver phosphorylase kinase is normal. Both forms of GSD IX have the normal blood sugar response to glucagon, whereas GSD VI does not. Equally, the glucagon tolerance curve is flat in GSD XI although in vitro activity of glycolytic enzymes is normal. The in vivo administration of glucagon in GSD XI is followed by the normal increase of both urinary 3'5'-AMP and hepatic phosphorylase activity. GSD V may have increased activity of muscle phosphorylase kinase. Deficiencies of debrancher, liver phosphorylase and liver phosphorylase kinase can occur singly or in combination. Before any novel treatment of GSD is initiated, one should obtain tissue for the biochemical determination of the exact type of GSD. This is so because the clinical signs may not indicate the type with the necessary precision, and because some types are compatible with normal life and thus may not require therapy, especially if the latter is unproved and potentially dangerous.  相似文献   

19.
In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.  相似文献   

20.
The translocation of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) to postsynaptic densities (PSDs) was investigated. When soluble CaM kinase II previously autophosphorylated was incubated with PSDs, the kinase was precipitated by centrifugation, indicating that the soluble kinase associated with PSDs and formed a PSD-CaM kinase II complex. Ca2+-independent activity generated by autophosphorylation of the kinase was retained in the complex. A number of PSD proteins were phosphorylated by the kinase associated with PSDs in both the absence and presence of Ca2+. When PSD-CaM kinase II complex was incubated at 30 degrees C, the enzyme was dephosphorylated and released from the complex. These results indicate that CaM kinase II reversibly translocates to PSDs in a phosphorylation-dependent manner.  相似文献   

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